Non-infected Cattle Research Articles

Evaluate the Concordance Between Two Diagnostic Methods, RT-PCR And ELISA Techniques for the Detection of CCHFV in Karbala City

Crimean-Congo haemorrhagic fever virus (CCHFV) is a widespread pathogen virus transmitted by ticks that causes a severe disease known as Crimean-Congo haemorrhagic fever in animals and humans. This study aims to investigate the occurrence of CCHFV in ticks and bovines in different regions of Karbala City/Iraq. Tick and blood samples were collected from four cattle farms, and molecular RT-PCR and serological analyses were conducted. The serum sodium concentration was calculated to compare infected and non-infected cattle with CCHFV. The study identified five tick genera, with Rhipicephalus (Boophilus) microplus and Amblyomma variegatum being the most prevalent species. However, no ticks tested positive for CCHFV. Serological analysis of the blood samples showed that none was positive for IgM antibody, but 19.00% tested positive for IgG antibodies and 29.3% for both IgG and IgM. The seroprevalence was higher in cattle from Ayn al-time (36.70%) and Hindiya (16.70 %) compared to other regions, the study found fair concordance between the two serological and molecular assays used for diagnosis of CCHFV virus; as well as the study concluded that sodium levels are an important prognostic factor for CCHFV infection, cattle with CCHFV.

Social network analysis reveals the failure of between-farm movement restrictions to reduce Salmonella transmission

An increasing number of countries are investigating options to stop the spread of the emerging zoonotic infection Salmonella Dublin (S. Dublin), which mainly spreads among bovines and with cattle manure. Detailed surveillance and cattle movement data from an 11-yr period in Denmark provided an opportunity to gain new knowledge for mitigation options through a combined social network and simulation modeling approach. The analysis revealed similar network trends for noninfected and infected cattle farms despite stringent cattle movement restrictions imposed on infected farms in the national control program. The strongest predictive factor for farms becoming infected was their cattle movement activities in the previous month, with twice the effect of local transmission. The simulation model indicated an endemic S. Dublin occurrence, with peaks in outbreak probabilities and sizes around observed cattle movement activities. Therefore, pre- and postmovement measures within a 1-mo time window may help reduce S. Dublin spread.

Rearing of Rhipicephalus annulatus ticks on rabbits for the biological transmission of Anaplasma marginale.

Anaplasma marginale is an obligate intraerythrocytic rickettsial parasite that infects cattle in tropical and subtropical regions. There is no evidence that A. marginale inoculation can be used to culture Rhipicephalus annulatus in rabbits. This study aimed to determine the molting of R. annulatus larvae, nymphs, and adults on rabbits as well as nymphs and adults of R. annulatus on calves with or without A. marginale. Transstadial, horizontal, and transovarial transmissions of A. marginale in R. annulatus reared on rabbits and calves were evaluated. Engorged female ticks were collected from field samples of A. marginale-infected and non-infected cattle. We divided the eight rabbits into two groups: A and B. Group A rabbits were infected with A. marginale through parenteral inoculation, whereas Group B rabbits were kept as a control. The "clean rabbits" in Group B were observed for tick rearing without A. marginale. Polymerase chain reaction was used to screen A. marginale in rabbits and stages of tick. The complete life cycle of R. annulatus with or without A. marginale was observed on rabbits. A 6.5-day longer life cycle was observed in ticks harboring A. marginale than in ticks without A. marginale. To observe transstadial transmission, transstadial, horizontal, and transovarial transmissions of A. marginale in R. annulatus ticks were experimentally observed in one clean calf fed separately with infected nymphs and female adult ticks. We experimentally observed transovarian, transstadial, and transovarial transmission of A. marginale in R. annulatus ticks as a biological vector reared on calves and rabbits. We used rabbits as a model animal for rearing R. annulatus ticks and culture of A. marginale.

Comparison of clinical, hematological and serum biochemical parameters between foot-and-mouth disease infected and non-infected Nigerian breeds of cattle.

Foot and mouth disease (FMD) still remains one of the major hindrances to livestock productivity in Africa as a whole and Nigeria in particular, with estimated well over 7billions losses to FMDV infection per annum in Nigeria. We therefore sought to establish and compare the clinical, hematological and biochemical indices in FMD infected and non-infected cattle. A total of 60 cattle including 40 apparently healthy and 20 FMD-infected cattle were sampled in Central Abattoir, Akinyele and Akufo Farm settlement, Ibadan, Oyo state, Nigeria respectively. Confirmatory diagnosis was done by accessing the presented pathognomonic clinical signs and the use of rapid diagnostic Antibody detection kit. Clinical parameters were recorded, and blood was drawn through the jugular vein for hematological and biochemical profiles. Standard hematological and serum biochemical techniques were used to analyzed the samples for hematological and biochemical parameters respectively. Results revealed higher clinical parameters in infected compared to the non-infected. All erythrocytic parameters were significantly lower in FMD infected cattle compared to non-infected cattle with P-values of 0.016, 0.015, 0.010, 0.001, 0.001 and 0.045 for PCV, Hb conc, RBC count, MCV, MCH and MCHC values respectively. Significant differences (P<0.05) of 0.001, 0.001, 0.001, 0.000, 0.001 and 0.001 for Lymphocyte, Neutrophil, Absolute lymphocyte, Absolute monocyte, Absolute neutrophil and platelets respectively. There were significant differences (P<0.05) of 0.009,0.001, 0.026, 0.001, 0.014, 0.001,0.001, 0.001, 0.001 and 0.001 for Total serum protein, Albumin, Globulin, Glucose, Creatinine, AST, ALT, ALP, Na+ and K+ respectively in infected cattle compared to non-infected cattle. We therefore conclude that, FMD-infected cattle showed lower clinical, hematological and serum biochemical parameters compared to uninfected cattle breeds in Nigeria.

Preferential differential gene expression within the WC1.1+ γδ T cell compartment in cattle naturally infected with Mycobacterium bovis.

Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis, continues to cause significant issues for the global agriculture industry as well as for human health. An incomplete understanding of the host immune response contributes to the challenges of control and eradication of this zoonotic disease. In this study, high-throughput bulk RNA sequencing (RNA-seq) was used to characterise differential gene expression in γδ T cells - a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. γδ T cell subsets are classified based on expression of a pathogen-recognition receptor known as Workshop Cluster 1 (WC1) and we hypothesised that bTB disease may alter the phenotype and function of specific γδ T cell subsets. Peripheral blood was collected from naturally M. bovis-infected (positive for single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA) and age- and sex-matched, non-infected control Holstein-Friesian cattle. γδ T subsets were isolated using fluorescence activated cell sorting (n = 10-12 per group) and high-quality RNA extracted from each purified lymphocyte subset (WC1.1+, WC1.2+, WC1- and γδ-) was used to generate transcriptomes using bulk RNA-seq (n = 6 per group, representing a total of 48 RNA-seq libraries). Relatively low numbers of differentially expressed genes (DEGs) were observed between most cell subsets; however, 189 genes were significantly differentially expressed in the M. bovis-infected compared to the control groups for the WC1.1+ γδ T cell compartment (absolute log2 FC ≥ 1.5 and FDR P adj. ≤ 0.1). The majority of these DEGs (168) were significantly increased in expression in cells from the bTB+ cattle and included genes encoding transcription factors (TBX21 and EOMES), chemokine receptors (CCR5 and CCR7), granzymes (GZMA, GZMM, and GZMH) and multiple killer cell immunoglobulin-like receptor (KIR) proteins indicating cytotoxic functions. Biological pathway overrepresentation analysis revealed enrichment of genes with multiple immune functions including cell activation, proliferation, chemotaxis, and cytotoxicity of lymphocytes. In conclusion, γδ T cells have important inflammatory and regulatory functions in cattle, and we provide evidence for preferential differential activation of the WC1.1+ specific subset in cattle naturally infected with M. bovis.

Study of the proviral load levels and mRNA expression of cytokines in peripheral blood mononuclear cells and somatic milk cells in cattle with different BLV infection profiles.

The retrovirus bovine leukemia virus (BLV) might produce abnormal immune function, associated with susceptibility to developing other infectious diseases, including mastitis. This study aimed to determine the proviral load and cytokines gene expression in peripheral blood mononuclear cells (PMBC) and milk somatic cells (SC) in BLV-infected and non-infected cattle. Of 27 BLV-infected cows in PBMC, 17 (62.96%) had a high proviral load (HPL), and 10 (37.04%) had a low proviral load (LPL). All SC samples had low proviral load (LPL-SC). Higher IFN-γ and IL-10 expression, and lower IL-12 and IL-6 expression, were found in PBMC from BLV-infected compared to BLV non-infected cattle. Moreover, higher IFN-γ, IL-12, and IL-6 expression, and lower IL-10 expression were observed in cattle with LPL-PBMC compared to HPL-PBMC. In milk samples, lower IFN-γ and higher IL-12 mRNA expression were observed in LPL-SC compared to BLV non-infected cattle in SC. IL-10 and IL-6 expression mRNA was significantly lower in LPL-SC than in SC from BLV non-infected cattle. This study shows that milk SC maintains lower proviral load levels than PBMC. This first report on Th1 and Th2 cytokines expression levels in SC may be relevant to future control strategies for BLV infection, mastitis, and udder health management.

Studies on haematological changes in GI nematodes in cattle and buffalo in Udaipur, district (Rajasthan)

A study on prevalence of for gastrointestinal nematode in the infected and non-infected cattle and buffalo of Udaipur district (Rajasthan) from Sept. 2020 to January 2021 by haematological parameters such as haemoglobin (Hb), packed cell volume (PCV), erythrocyte sedimentation rate (ESR), total leucocyte Count (TLC) and differential leucocyte count (DLC). The highly significant decrease in haemoglobin concentration innematode infected and non-infected buffalo (8.65±0.2335 and 10.12±0.1077) and (8.63±0.2886 and 09.84±0.1411) in infected and in non-infected cattle, respectively. The packed cell volume showed highly significant decrease in nematode infected and non-infected buffalo (28.98±0.2101 and 32.94±0.2477) and (27.64±0.5337 and 30.65±0.3956) in infected and non-infected cattle, respectively. The highly significant decrease in erythrocyte sedimentation rate in nematode infected and non-infected buffalo (0.80±0.0245 and 1.00±0.0167) and in cattle was found to be in infected (0.63±0.0287) and in non-infected (0.97±0.0240), respectively.Total leukocyte count (TLC) value significant increase for infected and noninfected buffalo and cattle buffalo (8.53±0.2784 and 10.45±0.2538) and (8.36±0.2582 and 10.49±0.1689), respectively. In the infected buffalo neutrophils (32.96±0.3084), eosinophils (5.50±0.2049).Basophils (0), lymphocytes (62.36±0.2948) and monocytes (5.48±0.1831). However, in non-infected buffalo neutrophils (37.21±0.3701), eosinophils (4.60±0.2196), basophils (0), lymphocytes (57.21±0.2802) and monocytes (4.99±0.2094). Whereas, in infected cattle neutrophils (28.02±0.2914), eosinophils (5.74±0.1942) basophils (0), lymphocytes (67.48±0.2979) and monocytes (5.57±0.1547). However, in non-infected cattle neutrophils (32.15±0.3088), eosinophils (3.88±0.1836) basophils (0), lymphocytes (61.59±0.3475) and monocytes (5.23±0.1832).There is reduction in haemoglobin, packed cell volume and ESR in the GI nematode infected cattle and buffalo. In total leukocyte count in buffalo and cattle lymphocytes, eosinophils and monocyte showed increased in infected group.

Seasonal prevalence of bovine trypanosomosis and trypanosome species distribution in Jimma Horo district, Oromia regional state, Western Ethiopia

A study was conducted to determine the prevalence of bovine trypanosomosis in rainy (June 2019) and dry (February 2020) seasons in Jimma Horoo district, Kellem Wollega Zone, Oromia Regional State, Ethiopia. A total of 720 blood samples were examined using buffy coat and thin blood smear techniques. The packed cell volume (PCV) of each animal was determined. The overall bovine trypanosomosis prevalence was 4.3% (χ2 = 1.25, P = 0.26). The prevalence was 5% (95% CI = 4.1–8.3) and 3.3% (95% CI = 2.7–6.3) in the dry and rainy reasons, respectively (P > 0.05). Trypanosoma congolense, T. vivax and T. b. brucei were detected in (60%), (33.3%) and (6.7%) of infected cattle, respectively. The highest trypanosome prevalence was observed in Burka Gudina (7.6%), and the lowest in Melka Nega village (2.1%). There were significant variations between trypanosome prevalence in relation to body condition scores (χ2 = 23.16; P = 0.0.00) of examined cattle. No significant difference (P > 0.05) was observed between seasons, age, and sex categories of cattle. The PCV values of trypanosome infected (22.94%) was significantly lower than non-infected cattle (26.47%) (χ2 = 19.60; (P < 0.05). The prevalence of bovine trypanosomosis in Jimma Horo district was low and it can be controlled by treatment of infected cattle using sanative pairs of trypanocidal drugs with additional application of deltamethrin pour-on in the dry season. Further, in view of poor sensitivity of buffy coat technique used the use of molecular techniques should be encouraged.

The effect of naturally acquired rumen fluke infection on animal health and production in dairy and beef cattle in the UK.

The incidence of paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, has greatly increased within Europe in the last 15–20 years. However, the production impacts of this disease are poorly understood. This study firstly aimed to investigate the prevalence of rumen fluke in England and Northern Ireland (NI) by conducting an abattoir survey of dairy and beef cattle which also allowed the impact of rumen fluke on carcass weight, conformation and fat classification to be assessed. Secondly, an experiment aimed to assess the impact of C. daubneyi infection on diarrhea score, production loss and welfare in dairy heifers, while also evaluating the impacts of treating infected heifers with oxyclozanide. Rumen fluke prevalence was greater in NI than in England, with 53.8% (95% CI 51.9 - 55.9%) of the NI cattle carcases sampled being infected compared to 16.3% (95% CI 15.8 - 16.8%) and 17.9% (95% CI 17.4 - 18.4%) detected at the two abattoirs in England. However, there was no significant difference (P > 0.05) in the cold carcass weight between infected and non-infected cattle. Similarly, carcass conformation and fat classification were unaffected (P > 0.05) by the presence of rumen fluke. In the second experiment, daily live weight gain (DLWG), diarrhea score and welfare score were also unaffected (P > 0.05) by rumen fluke infection and by oxyclozanide treatment against rumen fluke. The farms in this experiment were managed to a high standard and animals had no intercurrent disease. Therefore, these findings suggest that on well–managed farms, production losses (growth rates) should not be compromised as a result of sub-clinical rumen fluke infection.

High-resolution transcriptomics of bovine purified protein derivative-stimulated peripheral blood from cattle infected with Mycobacterium bovis across an experimental time course

ObjectivesImproved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response of Mycobacterium bovis-infected animals after bovine purified protein derivative (PPD-b) stimulation of whole blood—an important component of current bTB diagnostics—will provide new information for development of better diagnostics. MethodsRNA sequencing (RNA-seq) was used to study the peripheral blood transcriptome after stimulation with PPD-b across four time points (−1 wk pre-infection, and +1 wk, +2 wk, and +10 wk post-infection) from a 14-week M. bovis infection time course experiment with ten age-matched Holstein-Friesian cattle. ResultsIn vitro PPD-b stimulation of peripheral blood from M. bovis-infected and non-infected cattle elicited a strong transcriptional response. Comparison of PPD-b stimulated, and unstimulated samples revealed higher expression of genes encoding cytokine receptors, transcription factors, and interferon-inducible proteins. Lower expression was seen for genes encoding proteins involved in antimicrobial activity, C-type lectin receptors, inhibition of signal transduction, and genes encoding metal ion transporters. ConclusionsA transcriptional signature associated with the peripheral blood response to PPD-b stimulation consisting of 170 genes was identified exclusively in the post-infection time points. Therefore, this represents a panel of potential biomarkers of M. bovis infection.

Reliability of Some Clinical Parameters for Field Diagnosis of African Animal Trypanosomosis in Cattle

Presumptive and inaccurate diagnosis of cattle trypanosomosis among field veterinarians has led to misuse of trypanocides, development of drug resistance, toxicities and huge economic losses. This study assessed the reliability, specificity and sensitivity of some identified trypanosomosis associated signalments (anaemia using FAMACHA® guide, body condition score (BCS), superficial lymph nodes enlargement and jugular pulsation) as field diagnostic parameters. Blood and faecal samples were collected from 273 subjectively selected cattle for the determination of packed cell volume (PCV) and screening for trypanosome, and for helminth egg using McMaster technique, respectively. Data obtained from assessment of packed cell volume based on BCS, FAMANCHA score, superficial lymph nodes enlargement and jugular pulsation were analyzed using descriptive statistics and Chi square, while comparison of data using independent student ttest and one-way ANOVA was similarly conducted. Of the screened cattle, 16.5% (45/273) were positive for trypanosome. Single infection due to Trypanosoma spp. was found in 13.5% (37/273) cattle, while 2.9% (8/273) had trypanosome concurrently with Babesia spp. or helminth infections. The prevalence of Trypanosoma infection was higher in animals with mild or moderately pale mucous membrane, slight emaciation, palpable superficial lymph nodes and pulsating jugular vein. The mean PCV of Trypanosoma infected cattle (27.65±0.056) was lower than in non-infected cattle (31.30±0.36) (p&lt;0.05). Based on the jugular pulsation status, enlargement of the superficial lymph nodes, trypanosomosis state and Famancha category, there was no significant (p&lt;0.05) variation in the PCV of the examined cattle. A negative and low correlation (r = -0.054) existed between BCS and FAMACHA® anaemia score. Each of the clinical diagnostic parameters showed poor sensitivity when employed separately, but the sensitivity improved when applied together and showed 80% specificity to Trypanosoma infection. The results of the present study showed that Trypanosoma infected cattle were associated with anaemia, emaciation, weight loss, jugular pulsation and lymphadenophathy.&#x0D; Key Words: Trypanosomosis, Diagnosis, Cattle, Anaemia, Treatment

Babesiosis in cattle and ixodid tick distribution in Dasenech and Salamago Districts, southern Ethiopia

Babesiosis, caused by protozoan parasites of the genus Babesia, and ixodid ticks are considered to be one of the most important causes that result in significant economic losses in cattle production worldwide, including in Ethiopia. A cross-sectional study was undertaken in the Dasenech and Salamago Districts of South Omo zone Ethiopia to determine the prevalence and associated risk factors of babesia infection and ixodid ticks of cattle using Giemsa-stained thin and thick film techniques and morphological identification keys for babesia species and tick identification, respectively. Out of 470 examined cattle, 102 (21.7%) were infected by Babesiosis (15.53% Babesia bigemina and 6.17% Babesia bovis). A statistically significant association (p < 0.05) was observed between babesia infection and season and tick infestation. However, cattle that were infected with the Babesia parasite revealed a lower mean PCV value (21.49%) than noninfected cattle (28.29%) and showed a statistically significant (p < 0.05) difference with the occurrence of Babesia infection. The overall prevalence of ixodid ticks was 53.8% (253/470) and revealed a statistically significant association (p < 0.05) between the season and origin of the animal. However, no statistically significant association (p > 0.05) was observed between sex, age, and body condition score of the animal with the occurrence of ixodid tick. A total of 8040 adult ticks belonging to four tick genera, Amblyomma, Rhipicephalus (Boophilus), Hyalomma, and Rhipicephalus, were collected from various body parts and identified. The high prevalence of Babesia infection and ixodid ticks in cattle at the study sites requires seroprevalence and molecular studies to identify the predominant Babesia species and to detect Babesia in tick hemolymph for the identification of tick genera responsible for the occurrence of Babesia infection. Additionally, tailoring suitable and coordinated tick management methods using chemotherapy as well as strategic treatment to overt clinical cases of bovine babesiosis is critical.

Study on Prevalence of Trypanosomosis in Cattle of Sodo Zuriya District, Wolaita Zone, Southern Ethiopia.

Background Trypanosomosis is a significant impediment to Ethiopia's livestock and farm production, adding adversely to the overall growth of agriculture in general and the nation's food self-reliance efforts in particular. Methods Cross-sectional research was performed on the prevalence of trypanosomosis in cattle and the distinction of Trypanosoma species and potential factors in six selected provinces of the Sodo zuriya district in southern Ethiopia. Blood samples from the ear vein of 400 local and exotic cattle species were taken randomly. A hematocrit reader was used after centrifugation at 12,000 rpm for a five-minute assessment of anemic conditions of animals, and the anemia of the thin blood spraying and buffy coat procedure was tested to assess the incidence, detection, and packaging of Trypanosoma and the cell volume of each sampled animal. Results Accordingly, twenty cattle were positive for trypanosomosis, with an overall proportion of 5.0%. From this overall prevalence, Trypanosoma congolense (3.3%) and Trypanosoma vivax (1.8%) were the two common Trypanosoma species in this study. The highest and the lowest prevalence of trypanosomes occurred in Guttuto Larena (35%) and Dalbo Wogane (0.00%), respectively. Age-wise prevalence revealed that young adults (45%) were the most affected, followed by old adults (35%) and adults (20%). Cattle with poor body condition scores (65%) were the most affected, followed by cattle with medium (30%) and good (5%) body condition scores, and this was found to be statistically significant (p ≤ 0.002). In addition, the variation in packed cell volume (PCV) between infected and noninfected cattle was significantly different (p ≤ 0.0001). Conclusion Thus, the present study revealed the predominance of bovine trypanosomosis in the region and had a significant effect on body condition and anemia growth. The government and public should then collaborate in parasite-observed areas on the disease's management and prevention efforts in an environmentally sustainable way.

Haemoparasites in Cattle at Gbele Reserve, Upper West Region, Ghana

Haemoparasitic diseases is one of the major challenges that hamper cattle production in Africa with their effect usually manifested in production losses, late maturity, weight loss, still birth and increased susceptibility to other diseases. Information on the burden of some haemoparasites in wildlife/livestock interface in Ghana is scanty in literature. This study was carried out to determine the prevalence of haemoparasites in cattle at Gbele Reserve wildlife/livestock interface, Upper west region of Ghana. A total of 143 blood samples collected from four selected fringe communities namely Desima, Gbele, Wahabu and Sakalu were transported to Central Veterinary Laboratory at Pong-Tamale for analysis. Giemsa stained thin blood smears were examined for haemoparasites, and packed cell volume (PCV) for each sample was determined using Haematocrit method. Mean PCV value was 25% and 42% for infected and non-infected cattle respectively. An overall prevalence of haemoparasites was 2.8% for all samples examined. Village specific prevalence of 0.7%, 1.4%, 0.0% and 0.7% was recorded in cattle infected in Gbele, Desima, Sakalu and Wahabu respectively. The low prevalence of haemoparasites recorded was attributed to the earlier intervention by Pan-African Tsetse and Trypanosomosis Eradication campaign (PATTEC) project and the quarterly treatment regime employed by farmers. Babesia bovis and Anaplasma marginale were identified with prevalence of 2.1% and 0.7% respectively. The distribution of parasite based on sex was 2.1% for cows and 0.7% for bulls. Polymerase chain reaction and enzyme-linked immunosorbent assays are recommended alongside with thin blood smear examination in future studies for better sensitivity and identification of the haemoparasites to species level.

Bovine trypanosomosis and its vectors: prevalence and control operations in Kellem Wollega, Western Ethiopia

A cross-sectional study was conducted to estimate the prevalence of bovine trypanosomosis and to assess farmers’ perception of the disease and its control operations. From October to April 2012, a total of 586 cattle were sampled for the prevalence study. Buffy coat procedure and haematocrit value determination were performed. To capture the fly that was involved in the transmission dynamics, one hundred monopyramidal traps were deployed for 72 hours. A semi-structured questionnaire was conducted to study farmers’ perceptions of the diseases and their control operations. Trypanosomal infections were diagnosed in only 8.7 % (51) of animals. The overall prevalence of trypanosome infection in cattle was significantly varied between study districts (33.1% Dale Sadi and 66.9% Dale Wabera). Most infections were due to Trypanosoma congolense (81.8%) followed by T. vivax (15.6%) and mixed infections (2.6%). The association of hematological value changes and trypanosome infections was profound. The overall Packed Cell Volume (PCV) values of sampled cattle were 25.8%. A significant (P&lt; 0.05) variation in PCV values was recorded in infected (20.8%) and non-infected (26.5%) cattle. In the study period, a total of 2055 flies were captured and of which 92% belong to the genus Glossina followed by Stomoxys and Tabanids. Four types of tsetse species (G. pallidpes, G. m. morsitans, G. tachinoides, and G. f. fuscipes) were identified. The questionnaire survey revealed that trypanosomosis is the most important problem for agricultural activity and animal production in the study areas. Farmers are well aware of the problem, means of transmission, and the different control methods. Integrating tsetse control program with other trypanosomosis control options is recommended.

Novel polyprotein antigens designed for improved serodiagnosis of bovine tuberculosis

Recent studies have demonstrated potential for serologic assays to improve surveillance and control programs for bovine tuberculosis. Due to the animal-to-animal variation of the individual antibody repertoires observed in bovine tuberculosis, it has been suggested that serodiagnostic sensitivity can be maximized by use of multi-antigen cocktails or genetically engineered polyproteins expressing immunodominant B-cell epitopes. In the present study, we designed three novel multiepitope polyproteins named BID109, TB1f, and TB2f, with each construct representing a unique combination of four full-length peptides of Mycobacterium bovis predominantly recognized in bovine tuberculosis. Functional performance of the fusion antigens was evaluated using multi-antigen print immunoassay (MAPIA) and Dual Path Platform (DPP) technology with panels of monoclonal and polyclonal antibodies generated against individual proteins included in the fusion constructs as well as with serum samples from M. bovis-infected and non-infected cattle, American bison, and domestic pigs. It was shown that epitopes of each individual protein were expressed in the fusion antigens and accessible for efficient binding by the respective antibodies. The three fusion antigens demonstrated stronger immunoreactivity in MAPIA than that of single protein antigens. Evaluation of the fusion antigens in DPP assay using serum samples from 125 M. bovis-infected and 57 non-infected cattle showed the best accuracy (∼84 %) for TB2f antigen composed of MPB70, MPB83, CFP10, and Rv2650c proteins. Thus, the study results suggest a potential for the multiepitope polyproteins to improve diagnostic sensitivity of serologic assays for bovine tuberculosis.

Immunodiagnosis of cattle fascioliasis using a 27 kDa Fasciola gigantica antigen

Background and Aim:Diagnosis of fascioliasis depends on clinical symptoms and routine laboratory tests. Recently, antibodies and circulating antigens of Fasciola were used for detecting active infections. Therefore, this study aimed to identify Fasciola gigantica antigens in the sera of infected cattle using Western blotting and enzyme-linked immunosorbent assay (ELISA) for an accurate diagnosis of cattle infected with F. gigantica.Materials and Methods:Serum samples were obtained from 108, 23, and 19 cattle infected with Fasciola gigantica, Paramphistomum cervi, and Strongylids, respectively, including 57 non-infected cattle that were used as healthy cattle for the study. Western blotting and ELISA were then used to detect circulating Fasciola antigens at 27 kDa.Results:The target epitope was detected in an F. gigantica adult-worm antigen preparation, excretory/secretory products, and serum from cattle infected with F. gigantica. However, it was absent in sera from P. cervi, Strongylids, and healthy cattle. The purified 27 kDa F. gigantica (FPA-27) antigen was also detected in cattle serum using ELISA with high degrees of sensitivity and specificity (94% and 82%, respectively), and the area under the receiver operating characteristic curve was 0.89 with a highly significant correlation of p<0.0001.Conclusion:The FPA-27 is proposed to be a promising candidate for the serodiagnosis of fascioliasis in cattle.

Novel functional sequences uncovered through a bovine multiassembly graph

Many genomic analyses start by aligning sequencing reads to a linear reference genome. However, linear reference genomes are imperfect, lacking millions of bases of unknown relevance and are unable to reflect the genetic diversity of populations. This makes reference-guided methods susceptible to reference-allele bias. To overcome such limitations, we build a pangenome from six reference-quality assemblies from taurine and indicine cattle as well as yak. The pangenome contains an additional 70,329,827 bases compared to the Bos taurus reference genome. Our multiassembly approach reveals 30 and 10.1 million bases private to yak and indicine cattle, respectively, and between 3.3 and 4.4 million bases unique to each taurine assembly. Utilizing transcriptomes from 56 cattle, we show that these nonreference sequences encode transcripts that hitherto remained undetected from the B. taurus reference genome. We uncover genes, primarily encoding proteins contributing to immune response and pathogen-mediated immunomodulation, differentially expressed between Mycobacterium bovis-infected and noninfected cattle that are also undetectable in the B. taurus reference genome. Using whole-genome sequencing data of cattle from five breeds, we show that reads which were previously misaligned against the Bos taurus reference genome now align accurately to the pangenome sequences. This enables us to discover 83,250 polymorphic sites that segregate within and between breeds of cattle and capture genetic differentiation across breeds. Our work makes a so-far unused source of variation amenable to genetic investigations and provides methods and a framework for establishing and exploiting a more diverse reference genome.

Engineering Synthetic Lipopeptide Antigen for Specific Detection of Mycobacterium avium subsp. paratuberculosis Infection.

Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce glycopeptidolipids (GPL) on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-Valine-L-Isoleucine-L-Phenylalanine-L-Alanine, in which the N-terminal D-Phenylalanine is amido-linked with a fatty acid (C18–C20). The molecular and genetic characterization of this antigen demonstrated that L5P is unique to MAP. Knowledge of the structure of L5P enabled synthetic production of this lipopeptide in large quantities for immunological evaluation. Various studies described the immune response directed against L5P and confirmed its capability for detection of MAP infection. However, the hydrophobic nature of lipopeptide antigens make their handling and use in organic solvents unsuitable for industrial processes. The objectives of this study were to produce, by chemical synthesis, a water-soluble variant of L5P and to evaluate these compounds for the serological diagnosis of MAP using well-defined serum banks. The native L5P antigen and its hydrosoluble analog were synthesized on solid phase. The pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle. ROC analysis showed that L5P and also its water-soluble derivative are suitable for the development of a serological test for Johne's disease at a population level. However, these compounds used alone in ELISA have lower sensitivity (Se 82% for L5P and Se 62% for the water-soluble variant of L5P) compared to the Se 98% of a commercial test. Advantageously, these pure synthetic MAP specific antigens can be easily produced in non-limiting quantities at low cost and in standardized batches for robust studies. The fact that L5P has not been validated in the context of ovine paratuberculosis highlights the need to better characterize the antigens expressed from the different genetic lineages of MAP to discover new diagnostic antigens. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.

Alpha-2-Macroglobulin as a New Promising Biomarker Improving the Diagnostic Sensitivity of Bovine Paratuberculosis.

Johne's disease (JD) is a chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), which induces persistent diarrhea and cachexia. JD causes huge economic losses to the dairy industry due to reduced milk production and premature culling. Infected animals excrete MAP via feces during the prolonged subclinical stage without exhibiting any clinical signs. Therefore, accurate detection of subclinical stage animals is crucial for successful eradication of JD in the herd. In the current study, we analyzed serum samples of MAP-infected and non-infected cattle to identify potential biomarker candidates. First, we identified 12 differentially expressed serum proteins in subclinical and clinical shedder groups compared to the healthy control group. Second, we conducted ELISA for three selected biomarkers (alpha-2-macroglobulin (A2M), alpha-1-beta glycoprotein, and transthyretin) and compared their diagnostic performance with that of two commercial ELISA diagnostic kits. Serum A2M levels were significantly higher in the MAP-exposed, subclinical shedder, subclinical non-shedder, and clinical shedder groups than in the healthy control group, suggesting its possible use as a diagnostic biomarker for MAP infection. Furthermore, A2M demonstrated a sensitivity of 90.4%, and a specificity of 100% while the two commercial ELISA kits demonstrated a sensitivity of 67.83 and 73.04% and a specificity of 100%, respectively. In conclusion, our results suggest that measuring A2M by ELISA can be used as a diagnostic tool to detect MAP infection, considerably improving the detection rate of subclinical shedders and MAP-exposed animals that are undetectable using current diagnostic tools.